This proposal investigates the roles of bone morphogenetic proteins (BMPs) in chondrogenesis. Through analysis of mice lacking BMPR1A and/or BMPR1B, we demonstrated that signaling through these receptors is essential for chondrocyte proliferation, differentiation, and maintenance. These studies showed that BMPR1A and BMPR1B have overlapping functions at the condensation stage, but did not reveal the extent to which they collaborate at later stages. Moreover, while ActRI is insufficient on its own to support chondro- genesis beyond the condensation stage, the extent to which BMPR1A and/or BMPR1B alone can promote chondrogenesis is unknown. Resolution of this issue is important for future strategies targeted toward activation of specific BMP receptors in order to elicit defined responses in tissue engineering applications or in repair in vivo. We address these issues in Aim 1through characterization ActRI/Bmprla and ActRI/Bmprlb double mutants, and through the generation of mice in which loss of BMP receptor function can be induced at late gestation stages. It is likely that BMPs transduce the majority of their effects through Smads, but it is unknown whether or not all 3 BMP-specific Smads are required for chondrogenesis, or whether Smads have distinct vs. overlapping functions. We address this issue in aim two. Studies of Bmpr1a/1b compound mutants uncovered evidence that loss of BMP signaling is accompanied by increased output from FGF pathways. This finding suggests that BMP and FGF pathways act antagonistically during chondrogenesis, and raises the possibility that the relative balance between BMP and FGF signaling is a major determinant of chondrocyte progression through the growth plate. We test this and identify downstream components of FGF signaling pathways responsible for these effects in aim three through construction of mice in which relative output from BMP and FGF pathways is altered, and through the use of limb cultures. Analysis of BMP receptor mutants demonstrated that BMP pathways are essential for expression of Ihh, the key regulator of chondrocyte proliferation and differentiation in the growth plate. It has been shown in vitro that ERK1/2 antagonizes BMP signaling by directly phosphorylating, and thereby inactivating, BMP-specific Smads. In aim four, we test whether this mechanism is relevant to Ihh expression in the growth plate. Understanding how BMPs control distinct aspects of chondrogenesis and how FGFs antagonize these effects is vital to understanding how BMPs can be utilized most optimally in tissue engineering applications, for cartilage repair and maintenance, and potentially for treatment of chondrodysplasias caused by activating FGF mutations. University of California, Los Angeles David Geffen School of Medicine Department of Orthopaedic Surgery 2614 MacDonald Research Laboratories 675 Charles E Young Dr. South Los Angeles, CA 90095 PHS 398 (Rev.09/04) Page 2 Form Page 2 Principal Investigator/Program Director (Last, First, Middle): KEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name eRA Commons User Name Organization Karen Lyons LYONS2 UCLA Sean Brugger UCLA OTHER SIGNIFICANT CONTRIBUTORS Name Organization Akiko Hata Tufts University David Ornitz Washington University Lyons, Karen M information in the format shown below. Role on Project PI Postdoctoral fellow Role on Project Consultant Consultant Human Embryonic Stem Cells E3 No D Yes If the proposed project involves human embryonic stem cells, list below the registration number of the specific cell llne(s) from the following list: http://stemcells.nih.qov/reqistrv/index.asp. Usecontinuation pages as needed. If a specific line cannot be referenced at this time, include a statement that one from the Registry will be used. Cell Line Disclosure Permission Statement. Applicable to SBIR/STTR Only. See SBIR/STTR instructions. CHYes EH No PHS 398 (Rev. 09/04) Page 3 Form Page 2-continued Number the following pages consecutively throughout the application. Do not use suffixes such as 4a, 4b. Principal Investigator/Program Director (Last, First, Middle): Lyons, Karen M The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page 1 Description,